Opioid receptors are members of the large superfamily of G protein-coupled receptors (GPCRs), and are activated both by endogenously produced opioid neuropeptides and by a diverse group of addictive non- peptide drugs. Endogenous opioid peptides induce a complex series of regulatory processes, which begin with rapid phosphorylation and endocytosis of ligand-activated receptors. Non-peptide opioid drugs, including certain important opiate alkaloid such as morphine, have been observed to induce these regulatory processes to a smaller degree than opioid peptide agonists. Little is known about specific phosphorylation reactions occurring on the wild type receptor in intact cells. Furthermore, the functional effects of specific phosphorylation reactions on receptor regulation in physiologically relevant CNS neurons are not defined. The proposed studies address this question using novel protein biochemical, mass spectrometric and chemical genetic approaches. The Specific Aims are to 1) Define the effects of selected agonist ligands on regulatory phosphorylation of wild type receptors in intact cells;2) Define the functional significance of distinct phosphorylated receptor species to agonist-selective regulation of receptors;3) Elucidate a novel mechanism by which transient elevation of GRK2 activity produces a sustained increase in morphine- induced uOR endocytosis;and 4) Determine the importance of receptor regulatory mechanisms defined in HEK293 cells to morphine-induced endocytosis in cultured medium spiny neurons. These studies are specifically relevant to the fields of opioid biology and addiction research. They may also have more general implications for understanding ligand-dependent regulation of other GPCRs that, as a family, comprise the largest single group of clinically useful drug targets.